PANAGRELLUS REDIVIVUS ORNITHINE DECARBOXYLASE - STRUCTURE OF THE GENE, EXPRESSION IN ESCHERICHIA-COLI AND CHARACTERIZATION OF THE RECOMBINANT PROTEIN

A Southern blot analysis of the Panagrellus redivivus ornithine decarb oxylase (ODC) gene suggests that it is a single-copy gene that resides on a genomic 3.2 kb EcoRI fragment. Phage clones possessing ODC gene sequences were isolated from a genomic EMBL-4 library and purified. Th e phage DNA inserts were analysed and a 3.2 kb EcoRI fragment containi ng the entire ODC gene was isolated. The nucleotide sequence analysis of this fragment reveals that the gene is interrupted by two introns o f 47 and 49 bp. In the 5' non-translated region of the gene, putative API, VPE2 and c-Myc binding sites were identified. The ODC cDNA was ex pressed in a bacterial system as a His-fusion protein and the enzyme w as purified by Ni2+-chelating affinity chromatography. The subunit mol ecular mass, as deduced from the cDNA and shown by SDS/PAGE, is 47.1 k Da. On the basis of gel filtration analyses it is shown that the activ e enzyme is a dimer. The specific enzyme activity was determined to be 4.2 mu mol CO2/min/mg protein. The enzyme is dependent on pyridoxal 5 -phosphate as a cofactor, and the presence of dithioerythritol or othe r thiol-reducing agents is essential for maximal activity. The K-m val ue for L-ornithine was determined as 44 mu M. The K-i values for putre scine, alpha-difluoromethylornithine, alpha-hydrazino-ornithine and al pha-methylornithine were calculated as 51, 34, 0.34 and 42 mu M respec tively.